Development of constructs for recombinant expression of human follicle-stimulating hormone receptor in rod cells of the zebrafish

The follicle-stimulating hormone (FSH) is involved in the regulation of reproduction, by acting through a G protein-coupled receptor (GPCR) on the surface of target cells. Like most of the GPCRs, not much is known about the structure of the follicle-stimulating hormone receptor (FSHR). It is relatively difficult to purify the FSHR protein from gonadal tissues, due to its low abundance on its native cells, and therefore, to study its structure. For these reasons it is necessary to come up with a strategy that allows the production of large quantities of protein in order to use it in studies to obtain detailed structural information on full-length human FSHR. Via polymerase chain reactions, site-directed mutagenesis and other molecular biological methods, we were able to construct mutated receptors with different signal sequences and different tags, including the last fifteen amino acids of human rhodopsin which allow the receptor to be expressed on retinal rod cells of zebrafish. Mutations were designed in a manner that the receptor becomes inactive, i.e. incapable of signal transduction but still expressed at cell surface. The receptor constructs were first tested in human embryonic kidney cells (HEK 293-T) and their inserts will be used for transgenetic studies on zebrafish in the future.A hormona estimulante do folículo (FSH) está envolvida na regulação da reprodução, actuando através de um receptor acoplado a proteínas G (GPCR) localizado à superfície das células alvo. Tal como a maioria dos GPCRs, pouco se sabe acerca da estrutura do receptor da hormona estimulante do folículo (FSHR). Este receptor é relativamente difícil de purificar de tecidos gonadais, devido à pouca abundância com que se encontra nas suas células nativas, e, portanto, de estudar a sua estrutura. Por estas razões é necessário criar uma estratégia que permita a produção de grandes quantidades de proteína de modo a poder usá-la em estudos dirigidos à obtenção de informação detalhada da estrutura completa do FSHR. Através de reacções em cadeia da polimerase, mutagénese dirigida e outros métodos biomoleculares foi possível construir receptores mutantes com diferentes sequências sinal e diferentes tags, incluindo os últimos quinze aminoácidos da rodopsina humana que permitem a expressão do receptor em bastonetes da retina de peixe zebra. As mutações foram criadas de modo a que o receptor se torne inactivo, i.e. incapaz de realizar transdução de sinal mas que ainda é expresso à superfície da célula. Os receptores foram primeiro testados em células embriónicas humanas do rim (HEK 293-T) e os insertos serão usados em estudos de transgénese em peixe zebra

Avaliação do impacto das nanopartículas de prata no metabolismo celular: um estudo in vitro por metabolómica de RMN

Doutoramento em Nanociências e NanotecnologiaFace ao uso disseminado e enorme potencial terapêutico das nanopartículas de prata (AgNPs), o estudo dos seus efeitos biológicos é um assunto relevante e atual. O trabalho apresentado nesta tese teve como objetivo aprofundar o conhecimento existente sobre o impacto das AgNPs no metabolismo celular, usando a metabolómica por espectroscopia de ressonância magnética nuclear (RMN). Os tipos celulares escolhidos para este estudo foram queratinócitos da epiderme humana, células de hepatoma humano e macrófagos sanguíneos, por serem relevantes, respetivamente, ao nível da entrada, acumulação e captação de nanopartículas no organismo. O Capítulo 1 introduz as principais propriedades das AgNPs, a sua atividade biológica e potencial toxicidade, e descreve a abordagem metabolómica, incluindo uma breve revisão bibliográfica das suas aplicações em nanotoxicologia. O âmbito e os objetivos desta tese são, também, apresentados. O Capítulo 2 descreve os métodos experimentais utilizados ao longo deste trabalho, incluindo a caracterização das AgNPs, os procedimentos usados na cultura celular e nos ensaios biológicos, a colheita e preparação de amostras, a análise por RMN e o tratamento estatístico dos dados. No Capítulo 3, a atividade metabólica e a composição dos três tipos de células usados neste trabalho (queratinócitos HaCaT, células de hepatoma HepG2 e macrófagos RAW 264.7) são descritas com base na análise por RMN dos sobrenadantes dos meios de cultura (exometaboloma) e dos extratos celulares polares e lipofílicos (endometaboloma). O Capítulo 4 apresenta a análise metabolómica das células HaCaT expostas a AgNPs de diferentes tamanhos (10, 30 ou 60 nm de diâmetro) e revestimentos (citrato, polietilenoglicol ou albumina de soro bovino). Verificou-se que o metaboloma celular foi afetado mesmo a concentrações subtóxicas de AgNPs, sugerindo: aumento da glicólise e glutaminólise, alteração na atividade do ciclo dos ácidos tricarboxílicos (TCA) e nos processos de produção e transferência de energia, degradação de proteínas, síntese de glutationa (GSH), modificações a nível das membranas e do equilíbrio osmótico. Apesar de muitas variações serem comuns a todas as nanopartículas testadas, as AgNPs de 10 nm causaram os efeitos mais distintos, nomeadamente no que diz respeito à glicólise e à síntese/utilização de GSH. Além disso, a exposição celular a prata iónica (Ag+) confirmou o importante papel dos iões prata no mecanismo de ação das AgNPs, enquanto a comparação com o peróxido de hidrogénio (H2O2) permitiu destacar os efeitos relacionados com o stress oxidativo. No Capítulo 5 são apresentadas as respostas metabólicas das células de fígado HepG2 a dois tipos de AgNPs, umas obtidas por redução química e estabilizadas em citrato e as outras obtidas por síntese verde na presença de um extrato vegetal (Cit30 e GS30, respetivamente), ambas com centros metálicos de 30 nm. Os resultados sugeriram adaptações metabólicas em processos de produção de energia (metabolismo da glucose e sistema da fosfocreatina), autofagia e metabolismo lipídico, refletindo possivelmente a ativação de mecanismos de proteção. Ainda que os dois tipos de AgNPs tenham induzido muitos efeitos semelhantes, as Cit30 pareceram causar um maior impacto no ciclo TCA e na degradação de proteínas, enquanto as GS30 aparentaram induzir uma diminuição mais forte na síntese de fosfolípidos. A assinatura metabólica da prata iónica foi bastante semelhante à das AgNPs, sugerindo, no entanto, uma menor capacidade das células expostas a Ag+ extracelular para lidar com o stress oxidativo. O Capítulo 6 descreve a avaliação do impacto das AgNPs Cit30 no metaboloma de macrófagos de murganho RAW 264.7, a concentrações subtóxicas (decréscimos de ~5 e 20% na viabilidade celular). As alterações encontradas apontaram para: estimulação da glicólise (a baixa concentração de exposição), reprogramação do ciclo TCA (resultando numa intensa produção de itaconato e succinato e numa marcada depleção de ATP, consistentes com uma resposta pro-inflamatória), ativação da gluconeogénese, promoção da síntese de GSH e acumulação de creatina/fosfocreatina. Foram, ainda, observadas variações possivelmente relacionadas com a osmorregulação e a modificação membranar. De notar que os macrófagos expostos a Ag+ mostraram características semelhantes aos expostos a AgNPs (por ex., aumento da glucose intracelular – gluconeogénese), mas também revelaram efeitos distintos, nomeadamente em metabolitos envolvidos no ciclo TCA, em processos de transferência de energia e no metabolismo lipídico. Adicionalmente, viu-se que o metaboloma dos macrófagos respondeu de maneira diferente à exposição a H2O2 (por ex., tendência para diminuição da glicólise e sem efeitos observados na ativação da gluconeogénese ou da síntese de GSH), indicando que muitos dos efeitos induzidos pelas AgNPs não foram necessariamente mediados por stress oxidativo. Finalmente, com base na integração dos resultados apresentados ao longo dos capítulos anteriores, as principais conclusões deste trabalho são apresentadas e discutidas no Capítulo 7.The wide dissemination and promising therapeutic potential of silver nanoparticles (AgNPs) make the study of their biological effects a relevant upto- date subject. The work presented in this thesis aimed at deepening current understanding of the impact of AgNPs on cell metabolism, using NMR metabolomics of cultured mammalian cells. Epidermis keratinocytes, hepatoma cells and blood macrophages, relevant, respectively, to nanoparticle entry, accumulation and uptake, have been selected for the study. Chapter 1 introduces the main properties of AgNPs, including their biological activity and toxicological potential, and describes the metabolomics approach, briefly reviewing its applications in the field of nanotoxicology. Also, the scope and aims of this thesis are presented. Chapter 2 covers the experimental methods adopted during the course of this work, including the sources and characterisation of AgNPs, the procedures used in cell culture and biological assays, sample collection and preparation methods, NMR analyses and statistical data treatment. In Chapter 3, the metabolic activity and composition of the three cell types used in this work (HaCaT keratinocytes, HepG2 hepatoma cells and RAW 264.7 macrophages) are described based on the NMR analysis of culture medium supernatants (exometabolome) and polar/lipophilic cell extracts (endometabolome). Chapter 4 presents the metabolomic analysis of HaCaT skin cells exposed to AgNPs of different sizes (10, 30 or 60 nm in diameter) and coatings (citrate, polyethylene glycol or bovine serum albumin). The cellular metabolome was found to be affected even at sub-toxic concentrations of AgNPs, suggesting: upregulation of glycolysis and glutaminolysis, altered tricarboxylic acid (TCA) cycle activity, protein degradation, disruption of energy-producing pathways, glutathione (GSH) synthesis, membrane modification and changes in osmotic balance. Although several metabolic variations were common to all tested nanoparticles, the 10 nm AgNPs showed the most distinct effects, namely in regard to glycolysis and GSH synthesis/utilisation. Furthermore, cell exposure to ionic silver (Ag+) confirmed the major role of silver ions in AgNPs mode of action, while comparison with hydrogen peroxide (H2O2) allowed the effects related to oxidative stress to be highlighted. In Chapter 5, the metabolic responses of liver HepG2 cells to two types of 30 nm AgNPs, obtained by chemical reduction and stabilised with citrate or produced by green synthesis in the presence of a plant extract (Cit30 and GS30, respectively) are presented. Sub-toxic concentrations of AgNPs were proposed to induce metabolic adaptations in energy production processes (glucose metabolism and the phosphocreatine system), autophagy and lipid metabolism, possibly reflecting the activation of metabolism-mediated protective mechanisms. Although the two types of AgNPs induced many common effects, Cit30 appeared to have a greater impact on the TCA cycle and protein degradation, whereas GS30 seemed to induce stronger downregulation of phospholipid synthesis. The metabolic signature of Ag+ was largely similar to that of AgNPs, although suggesting a lower ability of cells exposed to extracellular Ag+ to cope with oxidative stress. Chapter 6 addresses the impact of Cit30 AgNPs on the metabolome of murine RAW 264.7 macrophages, at concentrations causing minimal (~5 and 20%) decreases in cell viability. Exposed cells were suggested to upregulate glycolysis (at the low exposure concentration), to reprogram the TCA cycle (resulting in marked production of itaconate and succinate and in ATP depletion, consistent with a pro-inflammatory response), to activate gluconeogenesis, to promote GSH synthesis, and to increase the creatine/phosphocreatine pool. Changes putatively related to osmoregulation and membrane modification were also observed. Notably, macrophages exposed to Ag+ showed common features to AgNPs-exposed cells (e.g. increased intracellular glucose suggesting gluconeogenesis), but also several distinct effects, for instance in metabolites involved in the TCA cycle, energy transfer processes or lipid metabolism. Furthermore, the cellular metabolome responded differently to H2O2 exposure (e.g. trend for downregulated glycolysis, no evidence of gluconeogenesis activation or of GSH upregulation), indicating that many of the AgNPs-induced effects were not necessarily mediated by oxidative stress. Finally, based on the integration of the results presented along the previous chapters, the main conclusions of this work are presented and discussed in Chapter 7

PEGylation-Dependent Metabolic Rewiring of Macrophages with Silk Fibroin Nanoparticles

Silk fibroin nanoparticles are emerging as promising nanomedicines, but their full therapeutic potential is yet to be realized. These nanoparticles can be readily PEGylated to improve colloidal stability and to tune degradation and drug release profiles; however, the relationship between silk fibroin nanoparticle PEGylation and macrophage activation still requires elucidation. Here, we used in vitro assays and nuclear magnetic resonance based metabolomics to examine the inflammatory phenotype and metabolic profiles of macrophages following their exposure to unmodified or PEGylated silk fibroin nanoparticles. The macrophages internalized both types of nanoparticles, but they showed different phenotypic and metabolic responses to each nanoparticle type. Unmodified silk fibroin nanoparticles induced the upregulation of several processes, including production of proinflammatory mediators (e.g., cytokines), release of nitric oxide, and promotion of antioxidant activity. These responses were accompanied by changes in the macrophage metabolomic profiles that were consistent with a proinflammatory state and that indicated an increase in glycolysis and reprogramming of the tricarboxylic acid cycle and the creatine kinase/phosphocreatine pathway. By contrast, PEGylated silk fibroin nanoparticles induced milder changes to both inflammatory and metabolic profiles, suggesting that immunomodulation of macrophages with silk fibroin nanoparticles is PEGylation-dependent. Overall, PEGylation of silk fibroin nanoparticles reduced the inflammatory and metabolic responses initiated by macrophages, and this observation could be used to guide the therapeutic applications of these nanoparticles. © 2019 American Chemical Society

Metabolic Signatures of Lung Cancer in Biofluids: NMR-Based Metabonomics of Blood Plasma

In this work, the variations in the metabolic profile of blood plasma from lung cancer patients and healthy controls were investigated through NMR-based metabonomics, to assess the potential of this approach for lung cancer screening and diagnosis. PLS-DA modeling of CPMG spectra from plasma, subjected to Monte Carlo Cross Validation, allowed cancer patients to be discriminated from controls with sensitivity and specificity levels of about 90%. Relatively lower HDL and higher VLDL + LDL in the patients' plasma, together with increased lactate and pyruvate and decreased levels of glucose, citrate, formate, acetate, several amino acids (alanine, glutamine, histidine, tyrosine, valine), and methanol, could be detected. These changes were found to be present at initial disease stages and could be related to known cancer biochemical hallmarks, such as enhanced glycolysis, glutaminolysis, and gluconeogenesis, together with suppressed Krebs cycle and reduced lipid catabolism, thus supporting the hypothesis of a systemic metabolic signature for lung cancer. Despite the possible confounding influence of age, smoking habits, and other uncontrolled factors, these results indicate that NMR-based metabonomics of blood plasma can be useful as a screening tool to identify suspicious cases for subsequent, more specific radiological tests, thus contributing to improved disease management.ERDF - Competitive Factors Thematic Operational ProgrammeFCT/PTDC/ QUI/68017/2006FCOMP-01-0124-FEDER-007439SFRH/BD/ 63430/2009National UNESCO Committee - L'Oréal Medals of Honor for Women in Science 200Portuguese National NMR Network - RNRM

Portuguese botanical gardens: before and after 2020

info:eu-repo/semantics/publishedVersio

Liver biopsy – evolution in recent cases

Os A.A. estudaram os resultados das biopsias hepáticas efectuadas no seu Serviço, com o objectivo de avaliar a evolução da prática relacionada com a sua realização. Identificaram as biopsias hepáticas realizadas nos Serviços de Medicina II, desde 1989 a 2001, na base de dados do Hospital, através da pesquisa no procedimento 50.11 (CID-9). Obtiveram assim um total de 610 biopsias no período considerado. Avaliaram com mais pormenor duas populações, uma constituída pelos episódios de 1991/ 1992 (n=66) – grupo A, confrontando-a com a de 2000/2001 (n=106) - grupo B. Após o levantamento dos processos clínicos, consideraram para estudo 48 doentes no grupo A (72,7 %) e 85 no grupo B (80,2 %), sendo os restantes excluídos por insuficiência de dados. Nestes grupos foram avaliados vários parâmetros clínicos e laboratoriais, sendo efectuadas comparações entre os grupos. Dos resultados, os A.A. destacam e comentam o aumento significativo de biopsias programadas em relação às executadas em doentes já internados, no grupo mais recente, e um maior número total e percentual de casos em que se optou pela alta no dia da execução do exame. O advento da radiologia de intervenção também se fez sentir neste trabalho, pela realização de biopsias guiadas por tomografia axial computorizada (TAC) em 12 % dos casos no segundo grupo, técnica inexistente no primeiro. Nas indicações, houve diminuição do peso da avaliação para diagnóstico/estadiamento de doença hepática alcoólica, enquanto se verificou aumento das biopsias para avaliação de alterações isoladas dos enzimas hepáticos, de massas hepáticas e no contexto do pré e pós-transplante hepático. Como diagnósticos estabelecidos com a ajuda desta técnica, destacamos a prevalência da esteato-hepatite não alcoólica no grupo mais recente.The A.A. analysed the results of liver biopsies performed in their Services, with the purpose of evaluating the evolution of the practice related to the performance of the diagnostic procedure. The liver biopsies performed in the Medicina 2 Services, from 1989 to 2001, were analysed. The procedure was identified in the Hospital data base using the code 50.11 (CID-9). There were 610 liver biopsies registered during the study period. Two populations were evaluated in greater detail. One comprising the cases from 1991/1992 (n=66) – group A -, comparing it with the second group from 2000/2001 (n=106) – group B. Several clinical and laboratorial parameters were analyzed in 48 patients in group A (72, 7 %) and 85 in group B (80, 2 %). From the results obtained, the authors point out and comment on the significant rise in programmed liver biopsies compared to those performed in inpatients, in the most recent group(B) and a larger number of cases that were discharged the same day of the procedure. The advent of interventional radiology was also noted in this paper; the performance of CAT scan guided liver biopsies in 12 % of the episodes in group B, as compared to none in group A. Where indications were respected, there were less procedures for diagnosis / staging of alcoholic liver disease and more to evaluate isolated altered liver enzymes, liver masses and control of pre and post hepatic transplant. From the diagnosis established with the aid of this technique, the authors highlight the prevalence of non alcoholic steatohepatitis in the most recent group

Treating Advanced Hepatocellular Carcinoma with Sorafenib: A 10-Year Single Center Experience

Introduction: Sorafenib was the first therapy used for systemic treatment of unresectable hepatocellular carcinoma (HCC). Multiple prognosis factors associated with sorafenib therapy have been described. Objectives: The aim of this work was to evaluate survival and time to progression (TTP) on HCC patients treated with sorafenib, and check for predictive factors of sorafenib benefit. Materials and Methods: Retrospectively, data from all HCC patients treated with sorafenib in a Liver Unit from 2008 to 2018 were collected and analyzed. Results: Sixty-eight patients were included; 80.9% were male, the median age was 64.5 years, 57.4% had Child-Pugh A cirrhosis and 77.9% were BCLC stage C. Macrovascular invasion (MVI) was present in 25% of the patients and 25% of the subjects had other extrahepatic metastasis. The median survival was 10 months (IQR 6.0–14.8) and median TTP was 5 months (IQR 2.0–7.0). Survival and TTP were similar between Child-Pugh A and B patients: 11.0 months (IQR 6.0–18.0) for Child-Pugh A and 9.0 months (IQR 5.0–14.0) for Child-Pugh B (p = 0.336). In univariate analysis, larger lesion size (LS >5 cm), higher alpha-fetoprotein (AFP >50 ng/mL), and no history of locoregional therapy were statistically associated with mortality (HR 2.17, 95% CI 1.24–3.81; HR 3.49, 95% CI 1.90–6.42; HR 0.54, 95% CI 0.32–0.93, respectively), but only LS and AFP were independent predictive factors, as shown in multivariate analysis (LS: HR 2.08, 95% CI 1.10–3.96; AFP: HR 3.13, 95% CI 1.59–6.16). MVI and LS >5 cm were associated with TTP shorter than 5 months in univariate analysis (MVI: HR 2.80, 95% CI 1.47–5.35; LS: HR 2.1, 95% CI 1.08–4.11), but only MVI was an independent predictive factor of TTP shorter than 5 months (HR 3.42, 95% CI 1.72–6.81). Regarding safety data, 76.5% of patients reported at least one side effect (any grade), and 19.1% presented grade III–IV adverse effects leading to treatment discontinuation. Conclusions: We observed no significant difference in survival or TTP in Child-Pugh A or Child-Pugh B patients treated with sorafenib, as compared to more recent real-life studies. Lower primary LS and AFP were associated with a better outcome, and lower AFP was the main predictor of survival. The reality of systemic treatment for advanced HCC has recently changed and continues to evolve, but sorafenib remains a viable therapeutic option